JP2023543810A - Activated soluble carriers for affinity binding and cell culture - Google Patents

Abstract

an activated soluble carrier comprising an ionotropically crosslinked compound comprising a polymeric material having at least one repeating unit comprising an ionically crosslinked carboxylic acid group and an activated hydroxyl group; wherein the hydroxyl group is activated by N,N'-disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidyl chloroformate in a solvent to form a succinimidyl carbonate group for ligand binding. form. Methods of forming activated soluble carriers, culturing cells on activated soluble carriers, and recovering cells from soluble carriers are provided.

Description

Cross-reference of related applications

Title 35 of the United States Code, United States Provisional Patent Application No. 63/084,153, filed September 28, 2020, the contents of which are relied upon and incorporated herein by reference in their entirety. Claiming the benefit of priority under Article 119 of the Patent Act.

The present disclosure generally relates to activated soluble carriers. In particular, the present disclosure relates to activated soluble carriers for use in cell culture and cell capture.

Cell therapy is gaining popularity as a treatment for many diseases. In traditional cell therapy production, the methods are labor intensive and formatted for production in small batches. However, many newly emerging cell therapies require isolation of low-frequency cells of interest from a heterogeneous cell mixture and subsequent large-scale expansion of the captured cells. Traditional small batch production techniques cannot meet the growing need for production methods for cell therapy that allow isolation of target cells and efficient culture of cells.

Traditional cell isolation techniques use ligand-receptor interactions to isolate cells and require the ligand to be immobilized on a solid support. However, traditional methods of immobilizing ligands on solid supports rely on activation of carboxylic acid groups, which can affect the mechanical integrity of the support and cause support degradation during further processing. There is sex.

Embodiments of the present disclosure provide activated soluble carriers. The carrier allows cell culture, cell capture, or both while maintaining the mechanical integrity of the cell culture medium. Dissolvable carriers do not require a polymeric adhesive coating since the ligands that enable cell capture or cell culture are covalently immobilized and thus permanently attached to the dissolvable carrier. Due to the nature of the ionic cross-links within the soluble carrier, the carrier maintains its mechanical integrity even in the presence of culture medium.

The carrier according to embodiments of the present disclosure allows for simplification of downstream processing, as the carrier is dissolvable and therefore can disappear on demand. Thus, soluble carriers provide improved recovery of captured or cultured cells since the carrier itself can be dissolved on demand, such as by digestion by addition of enzymes. In addition, the soluble carrier can be in any suitable format (e.g., beads, fibers, foam monoliths, among others), and is not limited to planar cell culture, such as two-dimensional (2D) monolayer cell culture. .

In one aspect, the activated soluble carrier comprises an ionotropically crosslinked compound comprising a polymeric material having at least one repeating unit comprising an ionically crosslinked carboxylic acid group and an activated hydroxyl group. , where the hydroxyl group is activated by N,N'-disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidyl chloroformate in a solvent to form a succinimidyl carbonate group for ligand binding. do.

In some embodiments, carboxylic acid groups are ionically crosslinked with polyvalent cations. In some embodiments, the solvent is an aprotic solvent. In some embodiments, the aprotic solvent is an anhydrous solvent.

In some embodiments, at least one repeating unit comprises:

In some embodiments, the polymeric material includes a polygalacturonic acid (PGA) compound. In some embodiments, the PGA compound comprises at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, or a salt thereof. In some embodiments, the partially esterified pectic acid comprises a degree of esterification from 1 mol% to 40 mol%. In some embodiments, the partially amidated pectic acid comprises a degree of amidation of 1 mol% to 40 mol%.

In some embodiments, the soluble carrier includes structures including beads, fibers, fabrics, foams, or coatings. In some embodiments, the soluble carrier includes porous beads. In some embodiments, the dissolvable carrier comprises a macroporous foam. In some embodiments, the soluble carrier comprises a coating for the cell culture surface of the cell culture vessel.

In some embodiments, soluble carriers are dissolved by digestion with enzymes, chelating agents, or a combination thereof. In some embodiments, the enzyme includes a non-proteolytic enzyme. In some embodiments, the non-proteolytic enzyme is selected from the group consisting of pectinolytic enzymes and pectinases. In some embodiments, digestion of the soluble carrier is completed in less than about 1 hour. In some embodiments, digestion of the soluble carrier is completed in less than about 15 minutes. In some embodiments, the ligand includes a protein, peptide, peptoid, saccharide, or drug.

In one aspect, a method of forming an activated soluble carrier includes forming a soluble carrier by adding a polymer solution containing a polygalacturonic acid (PGA) compound to a solution containing at least one polyvalent cation. the PGA compound is selected from at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof; and activating the hydroxyl groups to form succinimidyl carbonate groups on the PGA compound of the soluble carrier by adding an oxidation solution to form an ionotropically crosslinked compound.

In some embodiments, the method further includes rinsing the soluble carrier to remove unbound hydroxyl-containing compounds prior to activation. In some embodiments, the method further comprises attaching a ligand to the activated hydroxyl group to produce a ligand-soluble carrier conjugate. In some embodiments, ligands include proteins, peptides, peptoids, saccharides, and drugs. In some embodiments, the ligand soluble carrier conjugate comprises at least one unit comprising:

In some embodiments, the method further includes rinsing the activated soluble carrier with a solution containing at least one polyvalent cation after activation.

In some embodiments, ionotropically crosslinked compounds can be formed into structures including beads, fibers, fabrics, or foams. In some embodiments, the ionotropically crosslinked compound can be applied as a coating to a cell culture surface.

In some embodiments, the activation solution includes an activator and a solvent. In some embodiments, the activator comprises N,N'-disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidyl chloroformate. In some embodiments, the solvent includes an aprotic solvent.

In some embodiments, the polygalacturonic acid compound is pectic acid, partially esterified or amidated pectic acid having a degree of esterification or amidation from 1 to 40 mol%, or a salt thereof. Contains at least one of the following. In some embodiments, the polygalacturonic acid compound contains less than 20 mole percent methoxyl groups.

In one aspect, a method of culturing cells on a soluble carrier includes the steps of seeding the cells on the soluble carrier; and contacting the soluble carrier with a cell culture medium.

In some embodiments, seeding the cells on the soluble carrier includes adhering the cells to the surface of the soluble carrier. In some embodiments, cells aggregate within the pores of the soluble carrier to form spheroids.

In some embodiments, contacting the soluble carrier with the cell culture medium includes depositing the soluble carrier in the cell culture medium. In some embodiments, contacting the soluble carrier with the cell culture medium includes continuously passing the cell culture medium over the soluble carrier. In some embodiments, continuously passing the cell culture medium over the soluble carrier includes removing at least a portion of the cell culture medium from contact with the soluble carrier and removing the cell culture medium from contact with the soluble carrier. contacting the soluble carrier with fresh cell culture medium such that the volume of the culture medium is maintained substantially constant.

In one aspect, a method of recovering cells from a soluble carrier includes the steps of: digesting the soluble carrier by exposing the soluble carrier to an enzyme, a chelating agent, or a combination thereof; Sometimes including a step of harvesting the exposed cells.

In some embodiments, the soluble carrier is selected from at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof. It contains ionotropically cross-linked polygalacturonic acid compounds, and the enzymes include non-proteolytic enzymes. In some embodiments, the non-proteolytic enzyme is selected from the group consisting of pectinolytic enzymes and pectinases. In some embodiments, digesting the soluble carrier comprises exposing the soluble carrier to between about 1 U and about 200 U of enzyme. In some embodiments, digesting the soluble carrier comprises exposing the soluble carrier to between about 1 mM and about 200 mM of a chelating agent.

Fluorescent image of cells seeded on a carrier according to an embodiment of the present disclosure Fluorescent image of cells seeded on a carrier according to an embodiment of the present disclosure Fluorescence images of cells seeded in the negative control described in Example 5 Fluorescent image of cells captured on a carrier according to an embodiment of the present disclosure Fluorescence images of cells captured in the negative control described in Example 6

Embodiments of the present disclosure provide activated soluble carriers. The carrier allows cell culture, cell capture, or both while maintaining the mechanical integrity of the cell culture medium. Furthermore, the ligands that enable cell capture or cell culture are covalently immobilized and thus permanently attached to the soluble carrier. The carrier according to embodiments of the present disclosure allows for simplification of downstream processing, as the carrier is dissolvable and therefore can disappear on demand.

In one aspect of the invention, an activated soluble carrier is provided. Soluble carriers include soluble ionotropically crosslinked materials. In some embodiments, the soluble material is pectinic acid or a pectic acid derivative or Contains alginic acid derivatives.

Traditional activation methods rely on activation of carboxylic acid groups. In contrast to conventional activation methods, the hydroxyl activation methods in embodiments of the present disclosure do not affect the carboxyl (COOH) groups involved in ionotropic gelation. Thus, embodiments of the present disclosure preserve the mechanical integrity of the support and allow further processing without risk of degradation, since the density of ionic crosslinks does not change. In embodiments of the present disclosure, soluble carriers can be activated for further ligand binding for several months with minimal risk of loss of immobilization capacity due to the high stability of carbonate binding. Can be saved.

Conventional attachment of ligands to soluble pectinic, pectic, or alginic acid derivatives via amine functions is carried out by two main methods. The first conventional method relies on the activation of a carboxylic acid group, which is converted into an activated ester, such as an NHS ester, and the formation of an amide bond between the support and the immobilized ligand. Such carbodiimide-based chemistry is the chemistry of choice for polymers containing carboxylic acid groups, carboxymethylcellulose, alginic acid, hyaluronic acid, and polyacrylic acid, among others, and amine-containing compounds can be combined with alginate or pectin derivatives (both of which are numerous). is a gelling polymer with carboxyl groups). A second conventional method involves the use of periodic acid or periodate salts to generate aldehyde groups by controlling the oxidation of cis-diols on the backbone of the polysaccharide. Such methods are used to activate polysaccharides with carboxylic acid groups, such as biopolymers containing alginic acid or other carboxylic acids.

However, conventional techniques result in a significant loss of mechanical properties of the polymeric support. In the case of carbodiimide-mediated binding, the crosslink density is significantly reduced when the carboxylic acid groups involved in ionic crosslinking are consumed to form activated esters. As a result, the reduced crosslinking density reduces the mechanical properties of the support, making it unable to withstand agitation, flow of cell culture medium, or the flow required for affinity capture within a column format. In the case of aldehyde-activated supports, the introduction of aldehyde groups by controlling oxidation using periodate or periodic acid results in a significant decrease in the molecular weight of the polymer and some oxidative decarboxylation, resulting in its As a result, the mechanical resistance of the carrier can be significantly reduced. Therefore, conventional techniques cannot activate soluble carriers without reducing crosslink density and while providing acceptable dimensional stability.

The present disclosure relates to activated soluble ionically cross-linked carriers for affinity capture of cells, cell culture, or a combination thereof. Activated carriers can be used for immobilization of ligands and subsequent capture of molecules or cells. The carrier is particularly suitable for immobilizing ligands such as peptides and proteins and for carrying out cell sorting or cell culture. The activated carrier can be dissolved on demand. For example, carriers can be eliminated or dissolved on demand by simple addition of enzymes and/or chelating agents. Solubility is advantageous for recovery of molecules or cells captured by affinity. Complete lysis also facilitates purification of captured targets.

Methods according to embodiments of the present disclosure rely on activation of hydroxyl groups from ionically crosslinked soluble carriers. Activation is by N,N'-disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidyl chloroformate in the solvent, which is highly reactive toward amine nucleophiles and durable. Forms a succinimidyl carbonate group that is capable of forming certain carbamate bonds. In some embodiments, the solvent can be an anhydrous solvent.

In embodiments, the hydroxyl group is activated with N,N'-disuccinimidyl carbonate (DSC) or N-hydroxylsuccinimidyl chloroformate in an anhydrous solvent to form a succinimidyl carbonate group. Activation of such hydroxyl groups allows efficient immobilization of the ligand while preventing loss of mechanical integrity of the soluble carrier.

In embodiments, the soluble material is preferably made from ionically cross-linked (ionotropically cross-linked) alginate or pectin derivatives such as pectate and pectinate. Such physical crosslinking relies on the interaction of carboxylic acid groups with polyvalent cations such as calcium. Such physical crosslinks are reversible and crosslinks in the material can be broken by contacting with a chelating agent. In some embodiments, the material can be completely digested by adding enzymes. Among enzymes, pectinase and alginate lyase can be used to digest pectate or pectinate and alginate materials, respectively.

By using bonds on hydroxyl groups instead of carboxyl groups, soluble carriers according to embodiments of the present disclosure maintain high ionic crosslink density and avoid the risk of loss of mechanical integrity or geometry of the carrier. Exclude.

In one aspect, the polymeric material forming the activated soluble carrier comprises at least one unit comprising:

In another embodiment, the ligand soluble carrier conjugate comprises at least one unit comprising:

A ligand can be any synthetic or natural molecule that has at least one amino group and is capable of interacting with a target cell. Preferred ligands are proteins, peptides, peptoids, certain saccharides, and drugs, among others.

The activated ionically crosslinked carrier geometry can have any suitable geometry. In some embodiments, the activated ionically crosslinked carrier comprises beads, fibers, fabrics, or foam monoliths. In some embodiments, the carrier comprises macroporous foam or porous beads. In some embodiments, the soluble carrier can be configured to be placed within a cartridge or container for cell culture, and thus can be configured to fill the volume of the cartridge. As an example, if the dissolvable carrier is in the form of a macroporous foam piece, activation of this foam carrier will result in the foaming necessary to properly fit the volume of the cartridge in which the foam piece is placed. Body geometry is preserved.

In some embodiments, the soluble carrier is macroporous, allowing easy flow of liquid throughout the material with low backpressure, facilitating the separation process. Macroporous materials can be prepared by any suitable method such as foaming, gas foaming, and foaming aeration, among others.

Methods according to embodiments of the present disclosure are useful for attaching ligands to highly porous foams made of polysaccharides with COOH groups, such as pectin derivatives, despite the acidic nature of the foams. The method is advantageous for functionalizing highly porous foams that provide easy flow, but the low amount of solids present in the material (usually less than 2% by weight) and the hydrogel The inherent low modulus of elasticity due to its nature and hence its ability to absorb large amounts of water can lead to dimensional stability problems. Preparation of large separation columns remains difficult due to weak mechanical properties. Therefore, bonding chemistries that maintain high ionic crosslinking, such as those described in this disclosure, help maintain acceptable mechanical resistance and dimensional stability.

Any suitable polymer or biopolymer can be used in the soluble carrier and in the method of forming a soluble carrier according to embodiments of the present disclosure. In some embodiments, the biopolymer comprises a polysaccharide that is hydrophilic, non-cytotoxic, and stable in the culture medium. The soluble carriers described herein can include at least one ionotropically crosslinked polysaccharide. Generally, polysaccharides have beneficial attributes for cell culture applications. Polysaccharides are hydrophilic, non-cytotoxic, and stable in culture media. Examples include pectic acid or salts thereof, also known as polygalacturonic acid (PGA), partially esterified pectic acid or salts thereof, or partially amidated pectic acid or salts thereof. included. Pectic acid can be formed by hydrolysis of certain pectin esters. Pectin is a cell wall polysaccharide that has a structural role in plants in nature. Primary sources of pectin include citrus peels (eg, lemon and lime peels) and apple peels. Pectin is a predominantly linear polymer based on a 1,4-linked alpha-D-galacturonate backbone randomly interrupted by 1,2-linked L-rhamnose. Average molecular weight ranges from about 50,000 to about 200,000 Daltons.

Alginate is an example of a polysaccharide polymer material for forming a soluble carrier or cell culture scaffold. Alginate is a binary copolymer of 1-4 linked β-D mannuronic acid (M) and α-L-guluronic acid (G). The monomers are arranged in a block-like pattern along the chains, with divalent cations (Ca2+, Ba2+, Sr2+) preferentially binding to the G blocks of the alginate, forming bonds between adjacent alginate chains. Therefore, the stability of the crosslinked gel depends on the amount of G blocks.

In some embodiments, polygalacturonic acid (PGA) polymers are used to form soluble carriers or cell culture scaffolds. In the case of polygalacturonic acid or pectic acid, each monomer unit can potentially participate in ionic crosslinking, resulting in a highly crosslinked gel. The high cross-linking capacity makes PGA attractive in terms of mechanical properties and high stability, especially when exposed to high ionic strength media, such as those commonly encountered in cell culture. Furthermore, higher solids contents can be achieved using PGA since the viscosity of PGA solutions is usually lower than those made from alginate.

In embodiments, the soluble carrier comprises a polygalacturonic acid compound. In an embodiment, the polygalacturonic acid compound is at least one of pectic acid, partially esterified or amidated pectic acid having a degree of esterification or amidation of from 1 to 40 mol%, or a salt thereof. Including one.

Polygalacturonic acid results from the controlled hydrolysis of pectin, a cell wall polysaccharide that plays a structural role in plants. They are predominantly linear polymers based on a 1,4-linked alpha-D-galacturonate backbone randomly interrupted by 1,2-linked L-rhamnose. The average molecular weight is about 50,000 to about 200,000 Daltons. The two main sources of pectin are, for example, citrus peels (mainly lemons and limes) or apple peels, which can be obtained by their extraction.

The polygalacturonic acid chains of pectin can be partially esterified, and the methyl and free acid groups are partially or completely neutralized with monovalent ions such as sodium, potassium, or ammonium ions. Good too. Pectinic acid is a partially esterified polygalacturonic acid with methanol, and their salts are called pectinates. The degree of methylation (DM) of commercially available high methoxyl (HM) pectin is typically, for example, about 60 to about 75 mol%, while the degree of methylation of low methoxyl (LM) pectin is between intermediate values and Inclusive ranges can be from about 1 to about 40 mol%, from about 10 to about 40 mol%, and from about 20 to about 40 mol%.

In embodiments, the soluble carrier is preferably prepared from LM pectin. In embodiments, the polygalacturonic acid contains less than 20 mole percent methoxyl groups. In embodiments, the polygalacturonic acid has no or negligible methyl ester content, such as pectic acid. For simplicity, this disclosure refers to both pectinic acid, which has no or very few methyl esters, and low methoxyl (LM) pectin as PGA. For the same reason, if pectic acid or pectinic acid is amidated, the degree of amidation must be low enough to allow crosslinking by ionotropic gelation.

The polygalacturonic acid chains of pectin may be partially amidated. Pectin in which polygalacturonic acid is partially amidated can be produced, for example, by treatment with ammonia. Amidated pectin contains a carboxyl group (˜COOH), a methyl ester group (˜COOCH ₃ ), and an amidation group (—CONH ₂ ). The degree of amidation may vary, for example from about 10% to about 40% amidation.

According to embodiments of the present disclosure, the soluble carrier described herein can include a mixture of pectic acid and partially esterified pectic acid. Blends with compatible polymers can also be used. For example, pectic acid and/or partially esterified pectic acid can be mixed with other polysaccharides such as dextran, substituted cellulose derivatives, alginic acid, starch, glycogen, arabinoxylan, agarose, etc. Glycosaminoglycans such as hyaluronic acid and chondroitin sulfate, and various proteins such as elastin, fibrin, silk fibroin, collagen, and their derivatives can also be used. Water-soluble synthetic polymers can also be blended with pectic acid and/or partially esterified pectic acid. Exemplary water-soluble synthetic polymers include polyalkylene glycols, poly(hydroxyalkyl(meth)acrylates), poly(meth)acrylamides and derivatives, poly(N-vinyl-2-pyrrolidone), and polyvinyl alcohol. , but not limited to.

In some embodiments, the polygalacturonic acid compound is pectic acid, partially esterified or amidated pectic acid having a degree of esterification or amidation from 1 to 40 mol%, or a salt thereof. Contains at least one of the following. In some embodiments, the polygalacturonic acid compound contains less than 20 mole percent methoxyl groups.

In one aspect, a method of forming an activated soluble carrier includes forming an ionotropically crosslinked compound, the step comprising at least one step of forming a polymer solution containing a polygalacturonic acid (PGA) compound. forming a soluble carrier by adding the PGA compound to a solution containing pectic acid, partially esterified pectic acid, partially amidated pectic acid, and activating the hydroxyl groups in the PGA compound of the soluble carrier by adding an activating solution.

In some embodiments, activated soluble carriers can be formed into structures including beads, fibers, fabrics, or foams. In some embodiments, the activated soluble carrier can be applied as a coating to a cell culture surface.

Soluble carriers according to embodiments of the present disclosure may be crosslinked to prevent dissolution in cell culture media. In some embodiments, the method involves crosslinking a pectic acid derivative, such as PGA, by ionotropic gelation. Preferably, crosslinking is carried out by ionotropic gelation. Ionotropic gelation is based on the ability of polyelectrolytes to crosslink in the presence of multivalent counterions to form crosslinked hydrogels. Crosslinking can be performed by external ionotropic gelation. A variety of divalent cations can be used for crosslinking, including non-limiting examples of calcium, strontium, or barium.

In some embodiments, the activation solution includes an activator and a solvent. Any suitable activator can be used in methods according to embodiments of the present disclosure. In some embodiments, the activating agent is N,N'-disuccinimidyl carbonate (DSC). In some embodiments, the activating agent is N-hydroxysuccinimidyl chloroformate. Any suitable solvent can be used in methods according to embodiments of the present disclosure. In some embodiments, the solvent includes an aprotic solvent. Since aprotic solvents do not have O--H or N--H bonds, side reactions can be avoided. In some embodiments, the aprotic solvent includes an anhydrous solvent. Non-limiting examples of aprotic solvents include anhydrous acetone, anhydrous DMSO, anhydrous NMP, and anhydrous DMAc, among others. In some embodiments, the solvent is anhydrous DMSO. In some embodiments, the aprotic solvent is water-miscible. The water miscibility of the solvent prevents excessive shrinkage of the support during activation, such as when activation is performed on a macroporous support that can be used in a column format. In some embodiments, the method The method further includes rinsing the soluble carrier to remove unbound hydroxyl-containing compounds prior to activation. The support can be carefully washed prior to activation to remove any unbound hydroxyl-containing compounds that may react with the succinimidyl carbonate reagent and reduce activation of the PGA. Hydroxyl-containing compounds may be present in the PGA porous foam as blowing additives, such as, but not limited to, low molecular weight sugars, plasticizers such as glycerol, and surfactants. In embodiments, the rinsing step is performed with water, followed by rinsing with a dry solvent such as DMSO. The degree of water remaining in the material can affect the level of activation. Thorough washing with an anhydrous solvent increases the degree of activation.

In some embodiments, methods according to embodiments of the present disclosure further include a post-activation rinsing step. In some embodiments, the method further includes rinsing the activated soluble carrier with a solution containing at least one polyvalent cation after activation. In order to avoid reducing ionic cross-linking, the rinsing step after activation and binding of the ligand is preferably carried out using a solution containing at least one multiply charged ion, such as for example calcium. As a non-limiting example, a CaCl2 solution is a solution containing at least one multiply charged ion.

In some embodiments, the method further comprises attaching a ligand to the activated hydroxyl group to produce a ligand-soluble carrier conjugate. In some embodiments, ligands include proteins, peptides, peptoids, saccharides, and drugs. In some embodiments, the ligand soluble carrier conjugate comprises at least one unit comprising:

Cell Culture, Cell Capture, and Cell Recovery Recent studies have shown that the environment that cells experience in vivo is more accurately represented in 3D cell cultures, as opposed to two-dimensional (2D) or monolayer cultures. It has been suggested and demonstrated that cellular responses in 3D cultures are more similar to in vivo behavior than cellular responses in 2D cultures. The additional dimensionality of 3D culture affects the spatial organization of cell surface receptors involved in interactions with surrounding cells, causing physical constraints on the cells and thereby inhibiting signals from outside to inside the cells. They are thought to influence transmission and ultimately gene expression and cell behavior, leading to differences in cellular responses.

Cell culture techniques have been developed to simulate the natural 3D environment of cells. For example, some bioreactors include a support in the form of a fixed bed or a packed bed to promote cell attachment and proliferation. Another example of a 3D cell culture technique is a porous 3D matrix or scaffold that promotes the growth and proliferation of cultured cells within the pores and other interior spaces of the matrix. However, such techniques often use protease treatments to harvest cells, which exposes cells to harsh conditions that can damage cell structure and function. In addition, the use of protease treatment often results in only a limited amount of cell detachment. With fixed bed materials, the densely packed nature of the fixed bed material makes it more difficult to circulate the protease agent throughout the bed and increase the yield of cells recovered. Similarly, it can be difficult to circulate protease agents into the interior space of the 3D matrix, resulting in difficulty in removing cells during the harvesting process. This difficulty is further exacerbated by the presence of extracellular macromolecules secreted by cultured cells that serve to attach the cells to the surface of the fixed bed material or matrix. Therefore, conventional cell culture techniques have used mechanical force instead of or in combination with protease treatment to harvest cells. Such methods and systems for harvesting cells apply mechanical force to release cultured cells from fixed bed materials or 3D matrices. For example, the fixed bed material, 3D matrix, or larger system containing the fixed bed material or 3D matrix can be shaken or vibrated to release cultured cells. However, using mechanical force can cause physical damage to cultured cells, reducing the yield of cell culture.

According to embodiments of the present disclosure, methods of culturing or capturing cells on the soluble carriers described herein are also disclosed. In some embodiments, the method involves cell capture or cell culture of cell aggregates or spheroids in a soluble carrier. In some embodiments, the method includes culturing cells on a soluble carrier within a bioreactor system. Any type of cell can be cultured on the soluble carrier, including, but not limited to, immortalized cells, primary cultured cells, cancer cells, stem cells (eg, embryonic or induced pluripotent), and the like. The cells can be mammalian cells, avian cells, fish cells, etc. Cells include, but are not limited to, kidney, fibroblast, breast, skin, brain, ovary, lung, bone, nerve, muscle, heart, colorectal, pancreatic, immune (e.g., B cells), blood, etc. Can be any tissue type. Cells may be in any culture form, including dispersed (eg, freshly seeded), confluent, two-dimensional, three-dimensional, spheroids, and the like. Culturing the cells on the soluble carrier can include seeding the cells on the soluble carrier. Seeding the cells on the soluble carrier may include contacting the carrier with a solution containing the cells. Activated carriers according to embodiments of the present disclosure are customizable and can be functionalized with different components or compounds (eg, proteins, peptides) that promote cell adhesion. When the seeded cells are introduced into the functionalized carrier, the cells adhere to the surface of the functionalized carrier.

Cultivating cells on a soluble carrier can further include contacting the carrier with a cell culture medium. Generally, contacting the carrier with a cell culture medium involves placing the cells to be cultured on the carrier in an environment containing the medium in which the cells are to be cultured. Contacting the carrier with the cell culture medium comprises pipetting the cell culture medium onto the carrier, or submerging the carrier in the cell culture medium, or continuously passing the cell culture medium over the carrier. sell. Generally, as used herein, the term "continuous" refers to culturing cells with a consistent flow of cell culture medium into and out of the cell culture environment. Such passage of the cell culture medium over the carrier in a continuous manner involves depositing the carrier in the cell culture medium for a predetermined period of time and then removing at least a portion of the cell culture medium after a predetermined period of time and dissolving the cell culture medium. The method may include adding fresh cell culture medium such that the volume of cell culture medium in contact with the sexual carrier remains substantially constant. Cell culture media can be removed and replaced according to a predetermined schedule. For example, at least a portion of the cell culture medium can be removed and replaced every hour, or every 12 hours, or every 24 hours, or every 2 days, or every 3 days, or every 4 days, or every 5 days. .

Any cell culture medium that can support cell growth can be used. Cell culture media can include, for example, without limitation, sugars, salts, amino acids, serum (eg, fetal bovine serum), antibiotics, growth factors, differentiation factors, colorants, or other desired factors. Exemplary cell culture media include Dulbecco's Modified Eagle's Medium (DMEM), Ham's F12 Nutrient Mixture, Minimum Essential Medium (MEM), RPMI Medium, Iscove's Modified Dulbecco's Medium (IMDM), MesenCult™-XF Medium ( (commercially available from STEMCELL Technologies Inc.).

According to embodiments of the present disclosure, methods for harvesting cells from the soluble carriers described herein are also disclosed.

Dissolved carriers disclosed herein are described as soluble and insoluble. As used herein, the term "insoluble" is used to refer to a material or combination of materials that does not dissolve and remains cross-linked under conventional cell culture conditions, including, for example, cell culture media. As used herein, the term "soluble" also refers to a material or material that is digested when exposed to an appropriate concentration of enzymes and/or chelating agents that digest or degrade the material or combination of materials. used to refer to a combination of The soluble carriers described herein are optional for providing a protected environment for the culture of cells in which cell-to-cell interactions and formation of extracellular matrix (ECM) in a 3D manner are promoted. The carrier may be in any suitable form. Because soluble carriers can be completely digested, cells can be harvested without damaging the cells when using protease treatment and/or mechanical harvesting techniques. Carriers prepared according to embodiments of the present disclosure provide highly efficient release of cells captured or cultured on the carrier by lysis of the carrier using non-proteolytic enzymes, chelating agents, or both. enable.

In some embodiments, the soluble carriers described herein are digested when exposed to a suitable enzyme that digests or degrades the material. The methods of harvesting cells described herein can include digesting the soluble carrier by exposing it to an enzyme. Non-proteolytic enzymes suitable for carrier digestion, cell harvesting, or both include pectinolytic enzymes or pectinases, a heterogeneous group of related enzymes that hydrolyze pectin materials. Pectinases (polygalacturonases) are enzymes that break down complex pectin molecules into shorter molecules of galacturonic acid. Commercially available sources of pectinase are generally Pectinex™ ULTRA SP-L (North Carolina, USA), which are pectinolytic enzyme preparations produced from selected strains of Aspergillus aculeatus. (commercially available from Novozyme North American, Inc., Franklinton). Pectinex™ ULTRA SP-L mainly contains polygalacturonase (EC3.2.1.15), pectin transeliminase (EC4.2.2.2), and pectinesterase (EC:3.1). .1.11). The EC designation is the Enzyme Commission's classification scheme for enzymes based on the chemical reaction they catalyze.

Exposing the soluble carrier to an enzyme can include exposing the carrier to an enzyme concentration of between about 1 and about 200 U. For example, the method exposes the carrier to an enzyme concentration of between about 2 U and about 150 U, or between about 5 U and about 100 U, or even between about 10 U and about 75 U, and all values therebetween. may include doing.

The methods of harvesting cells described herein can further include exposing the material to a chelating agent. In some embodiments, the soluble carriers described herein are digested upon exposure to a suitable chelating agent that digests or degrades the material. According to embodiments of the present disclosure, digestion of the soluble carrier comprises exposing the carrier to a divalent cation chelating agent. Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), ethylene glycoltetraacetic acid (ETGA), citric acid, and tartaric acid.

Exposing the soluble carrier to a chelating agent can include exposing the carrier to a chelating agent concentration of between about 1 mM and about 200 mM. For example, the method comprises introducing a carrier to a chelating agent concentration of between about 10 mM and about 150 mM, or between about 20 mM and about 100 mM, or even between about 25 mM and about 50 mM, and all values therebetween. may include exposing.

The time to complete digestion of the soluble carriers described herein can be less than about 1 hour. For example, the time to complete digestion of the carrier is less than about 45 minutes, or less than about 30 minutes, or less than about 15 minutes, or between about 1 minute and about 25 minutes, or between about 3 minutes and about 20 minutes, or It may even be between about 5 minutes and about 15 minutes.

Embodiments of the present disclosure are further described below with respect to certain illustrative and specific embodiments thereof, which are intended to be illustrative only and not limiting.

Example 1
Example 1 compares conventional ligand binding methods and binding methods according to embodiments of the present disclosure, as shown in Scheme I and Scheme II below. Scheme I is an example of a conventional or traditional method of attaching a ligand to a material such as alginate or polygalacturonic acid. Scheme II is an example of a method of attaching a ligand according to an embodiment of the present disclosure.

Scheme I: Activation and conjugation of polygalacturonic acid via carboxylic acid groups by traditional methods

Scheme II: Activation and attachment via hydroxyl groups of a soluble carrier (polygalacturonic acid) in embodiments according to the present disclosure

As shown in the traditional method shown in Scheme I, a carboxylic acid group is activated and then a ligand is attached to such activated carboxylic acid group (NHS ester). When derivatized carboxylic acids are converted to amide linkers, they are no longer able to contribute to ionic crosslinking with calcium ions as free carboxylic acids can. Therefore, the crosslink density decreases, resulting in a deterioration of the mechanical properties of the soluble carrier.

In contrast, as shown in the method according to an embodiment of the present disclosure shown in Scheme II, the ligand is attached to the hydroxyl group while the carboxylic acid group remains intact. Thus, the carboxylic acid groups remain available for ionic crosslinking by polyvalent cations such as calcium. Therefore, regardless of the amount of bound ligand, the crosslinking density does not decrease and the mechanical properties of the soluble carrier hardly change.

Example 2
Example 2 demonstrates the activation of a macroporous PGA-based dissolvable foam. A macroporous carrier was prepared as follows.

A 2% by weight PGA solution, approximately 162 g, was prepared by dissolving an appropriate amount of polygalacturonic acid sodium salt (PGA), available from Sigma Aldrich, in demineralized water using an oil bath (set temperature 104 °C). was prepared. The resulting solution was cooled to room temperature. To this solution, 0.97 g of Pluronic 123 was added and dissolved at low temperature while mixing. The resulting solution was then placed in a mixer bowl (eg, KitchenAid mixer bowl). Next, 17.5 g sucrose and 7.5 g glycerol were added to the bowl and the mixture was mixed gently until the sugar was completely dissolved. A solution prepared from 0.97 g of 150 kDa dextran and 0.125 g of Tween 20 in 10 ml of water was added to the bowl and mixed gently to achieve a homogeneous mixture. A dispersion consisting of 0.53 g CaCO3 (Sigma), 0.248 g sodium dodecyl sulfate, and 14.5 ml ultra pure (UP) water was then added to the bowl. The foam was prepared by whisking the suspension to incorporate air using a wire loop whisk at speed 2 for 5 minutes. Then, a freshly made glucono delta-lactone (GDL) solution prepared by mixing 3.77 g GDL and 12.6 g DMSO was quickly added to the bowl and continued whisking for about 60 seconds. The foam was left uncovered in a bowl on a bench at 23° C. for 3 hours to complete crosslinking. Next, the crosslinked foam was punched out into disks (slices) and sliced.

The foam pieces were then frozen at -80°C for 16 hours and then freeze-dried at -86°C and 0.11 mbar (11 Pa) for 72 hours. The resulting foam exhibits a dry foam density (DFD) of approximately 0.04-0.045 g/cm ³ .

The foam slices were then activated by reacting with DSC as follows. Briefly, 10 slices of 34 mg each were added to a 50 ml plastic tube and rinsed twice with UP water and three times with anhydrous DMSO to remove unbound material. Approximately 90% of the additive material is removed from the foam. The supernatant was then discarded and approximately 40 ml of a solution prepared by mixing 40 ml of anhydrous DMSO, 394 mg of N,N'-disuccinimidyl carbonate (DSC), and 167 mg of 4-dimethylaminopyridine (DMAP) added to the slice. The tube was placed on a shaker and stirred at room temperature (RT) for 5.5 hours. Slices were rinsed twice with 4 wt% CaCl ₂ , pH 8.5 to remove unreacted reagents. The foam can be lyophilized in this step and stored using a desiccant in the dark at 4° C. for several months.

Example 3
Example 3 shows the conjugation of the VN peptide on a soluble carrier derived from Example 2.

Per foam slice, 4 ml of a 5 mg/ml vitronectin-NH2 peptide (American Peptide Company, Inc.) solution in 4% CaCl2 adjusted to pH 9 was added. Slices were incubated in the oven at 60°C overnight. The slices were thoroughly rinsed with UP water and the amount of immobilized VN peptide was quantified using the bicinchoninic acid assay (BCA assay). BCA analysis showed that approximately 0.5 μg of peptide was immobilized per mg of dry foam. This foam withstood extensive rinsing without showing any signs of deterioration.

Example 4
Example 4 shows the immobilization of protein A onto an activated soluble carrier prepared as described in Example 2, except that 130 mg DSC and 62 mg DMAP were used and activation was carried out for 18 hours. to show the

For each foam slice prepared according to Example 2, 4 ml of a 0.25 mg/ml Protein A solution in 4% by weight CaCl2, pH 9 was added. Slices were incubated for 48 hours at RT and rinsed thoroughly once with 1% by weight Tergitol™ NP40 in Dulbecco's phosphate-buffered saline (DPBS) solution and then three times with DPBS. The amount of immobilized Protein A was quantified using the bicinchoninic acid assay (BCA assay). BCA analysis showed that approximately 0.9 μg of protein A was immobilized per mg of dry foam. The foam withstood extensive cleaning.

To demonstrate that the immobilized Protein A is functional, OKT3 mouse monoclonal antibody was conjugated to the PA functionalized foam. In a typical experiment, a solution containing 160 μg of OKT3 antibody in approximately 8 ml of DPBS was prepared. 4 ml of OKT3 antibody solution was added to each PA-functionalized foam slice. Slices were left at RT for 1 hour, then rinsed with 2 ml of 1% Tergitol NP40 in DPBS, followed by three rinses with 2 ml of DPBS. Elisa assay showed that approximately 0.75 μg of OKT3 antibody was immobilized per slice.

Example 5
Example 5 shows the culture of HEK293T cells in the porous structure of a foam functionalized with activated vitronectin peptide (VN) according to the embodiment described in Example 3. Such VN peptide-functionalized foams are referred to as "VN" within Example 5.

As a negative control, slices were activated according to the embodiment described in Example 2, but instead of being functionalized with vitronectin, they were blocked with ethanolamine. Such blocked foam is referred to as "EA" within Example 5.

Slices of both VN and EA functionalized foam prepared above according to Example 3 were cleaned as follows. Slices were placed into the wells of a polystyrene 6-well plate. 4 ml of 70% ethanol aqueous solution was added to each well and disinfected for 10 minutes. The aqueous ethanol solution was then removed by suction and the foam was rinsed three times with UP water.

After removing excess water, place the wet foam slices in a Costar® 6-well transparent flat-bottom ultra-low attachment surface multiwell plate, product number 3471, containing 4 ml of complete Iscove's modified Dulbecco's medium (IMDM). Corning Incorporated, Corning, New York, USA). Complete IMDM medium was prepared by mixing 450 ml IMDM, 50 ml FBS, 5 ml penicillin/streptomycin, and 5 ml Glutamax™ (commercially available from Thermo Fisher Scientific).

Once the foam slices absorbed the medium, excess medium was removed by aspiration. Next, 150 μl of medium containing 300K HEK293 T cells was added to each slice. The plate was left standing in an incubator at 37°C/5% CO2 for 3 hours to allow cells to adhere. Next, 4 ml of complete medium was added and cells were allowed to grow for 5 days.

One day after seeding, cell adhesion was evaluated by visual inspection using a fluorescence microscope after calcein staining. Cell counts were performed 5 days after seeding by digesting the foam scaffolds by adding a solution consisting of 0.5 ml trypsin, 2 ml pectinase 50 U, and 5 mM EDTA to each slice. It took about 5 minutes to dissolve.

Figure 1 shows the spreading and adhesion of HEK293 T cells seeded on VN-functionalized scaffolds according to Example 5 (VN). Figure 2 shows cells covering the entire surface of the scaffold 5 days after seeding. Counts showed that the cells had grown 6.5 times.

Figure 3 shows the fluorescence image of the negative control. In contrast to the adhesion shown in Figures 1 and 2, cells fail to adhere to negative control foam blocked with ethanolamine (EA). Instead, the cells were unable to adhere to the EA-blocked control foam and aggregated into clusters in FIG. 3.

Example 6
Example 6 demonstrates the capture of Jurkat cells expressing CD3 antigen by a foam scaffold functionalized with OKT3 antibody.

OKT3-conjugated foam slices prepared according to Example 4 were incubated with 450 ml Roswell Park Memorial Institute Medium (ATCC Modified RPMI), 50 ml FBS, 5 ml Penicillin/Streptomycin, and 5 ml Glutamax™ (from Thermo Fisher Scientific). (commercially available) into the wells of a Costar® 6-well clear flat bottom ultra-low attachment surface multiwell plate, product no. 3471 (Corning Incorporated, Corning, NY, USA). Moved. Excess medium was removed as much as possible by aspiration. Next, 150 μl of Jurkat suspension, 10,000 K (10 million)/ml, was added to each scaffold piece. Scaffolds were incubated at 37°C for 30 minutes to allow cell capture. The foam was then washed three times with D-PBS buffer after staining the cells with calcein. The stained cells were imaged. A large number of cells were captured by the OKT3-conjugated scaffold, as shown by the stained cells in Figure 4.

A protein A-functionalized scaffold without OKT3 was used as a negative control (referred to as "PA" within Example 6). Cells were stained with calcein and imaged. The images in Figure 5 show that, in contrast to Figure 4, only a small amount of cells were captured non-specifically with the negative control (PA-functionalized carrier without OKT3).

Comparative example 1
Comparative Example 1 shows that activation of carboxylic acid groups can have a negative effect on crosslinking and stability of the soluble carrier.

Foam slices were prepared using the method described in Example 2 as follows. A 2% by weight PGA solution, approximately 162 g, was prepared by dissolving an appropriate amount of polygalacturonic acid sodium salt (PGA), available from Sigma Aldrich, in demineralized water using an oil bath (set temperature 104 °C). was prepared. The resulting solution was cooled to room temperature. To this solution, 0.97 g of Pluronic 123 was added and dissolved at low temperature while mixing. The resulting solution was then placed in a mixer bowl (eg, KitchenAid mixer bowl). Next, 17.5 g sucrose and 7.5 g glycerol were added to the bowl and the mixture was mixed gently until the sugar was completely dissolved. A solution prepared from 0.97 g of 150 kDa dextran and 0.125 g of Tween 20 in 10 ml of water was added to the bowl and mixed gently to achieve a homogeneous mixture. A dispersion consisting of 0.53 g CaCO3 (Sigma), 0.248 g sodium dodecyl sulfate, and 14.5 ml ultra pure (UP) water was then added to the bowl. The foam was prepared by whisking the suspension to incorporate air using a wire loop whisk at speed 2 for 5 minutes. Then, a freshly made glucono delta-lactone (GDL) solution prepared by mixing 3.77 g GDL and 12.6 g DMSO was quickly added to the bowl and continued whisking for about 60 seconds. The foam was left uncovered in a bowl on a bench at 23° C. for 3 hours to complete crosslinking. Next, the crosslinked foam was punched out into disks (slices) and sliced. The foam pieces were then frozen at -80°C for 16 hours and then freeze-dried at -86°C and 0.11 mbar (11 Pa) for 72 hours. The resulting foam exhibits a dry foam density (DFD) of approximately 0.04-0.045 g/cm ³ .

In Example 2, the prepared foam slices were then activated with DSC. In contrast, the foam slices prepared in Comparative Example 1 were instead activated by reaction with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)/N-hydroxysuccinimide (NHS). .

Activation of EDC/NHS was performed as follows. The foam slices were rinsed four times with 4 ml of UP water to remove unbound material. The scaffold was then activated by adding 4ml of 200mM EDC and 50mM NHS. Activation was performed for 30 minutes at RT, and the scaffolds were then rinsed once with Dulbecco's phosphate buffered saline (DPBS). Excess DPBS was carefully removed by aspiration.

Protein A was then immobilized using the conditions described in Example 4.

By changing the activation reaction and activator, the foam slices prepared in Comparative Example 1 have activated carboxyl groups instead of having activated hydroxyl groups as described in Example 2. In contrast to Example 2, the foam slices obtained in Comparative Example 1 disintegrated during the rinsing process. This proves that the consumption of carboxyl groups to generate amide bonds negatively affects the stability of the foam due to a decrease in the density of ionic crosslinking sites.

It will be appreciated that the various disclosed embodiments may include particular features, elements, or steps described in connection with that particular embodiment. Additionally, although particular features, elements, or steps are described in connection with one particular embodiment, they may be replaced or combined with alternative embodiments in various combinations or permutations not illustrated. will also be recognized.

As used herein, the terms "the", "a", or "an" mean "at least one" and are limited to "only one" unless expressly indicated to the contrary. It should be understood that this should not be done. Thus, for example, reference to "an aperture" includes embodiments having more than one such "aperture" unless the context clearly indicates otherwise.

All scientific and technical terms used herein have meanings commonly used in the art, unless specified otherwise. The definitions provided herein are to facilitate understanding of certain terms frequently used herein and are not meant to limit the scope of the disclosure.

As used herein, "having", "having", "comprising", "comprising", "comprising", "comprising", etc. are used in an open-ended sense. and generally means "including, but not limited to."

Ranges can be expressed herein as from about one particular value, and/or to about another particular value. When such a range is expressed, examples include from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, eg, by use of the antecedent "about," it will be understood that the particular value forms another aspect. Furthermore, it will be appreciated that each endpoint of the range is significant both in relation to the other endpoints and independently of the other endpoints.

All numerical values expressed herein, unless specifically stated otherwise, should be construed as including "about" whether or not so stated. However, it is further understood that each recited numerical value is contemplated with the same precision whether or not the value is expressed as "about." Thus, both "dimensions less than 10 mm" and "dimensions less than about 10 mm" encompass embodiments of "dimensions less than about 10 mm" as well as "dimensions less than about 10 mm."

Unless otherwise specified, any method described herein is in no way intended to be construed as requiring its steps to be performed in a particular order. Thus, either the method claim does not actually recite the order in which the steps are to be followed, or it is not specifically stated in the claim or specification that the steps are to be limited to a particular order. No particular order of events is intended to be inferred in any way.

Various features, elements, or steps of a particular embodiment may be disclosed using the transitional phrase "comprising," but are not disclosed using the transitional phrase "consisting of" or "consisting essentially of." It should be understood that alternative embodiments are implied, including those that may be described as follows. Thus, for example, implicit alternative embodiments of a method comprising A+B+C include embodiments where the method consists of A+B+C, and embodiments where the method essentially consists of A+B+C.

Exemplary Implementations The following is a description of various aspects of implementations of the disclosed subject matter. Each aspect may include one or more of the various features, properties, or advantages of the disclosed subject matter. The implementations are intended to describe several aspects of the disclosed subject matter and are not to be considered as comprehensive or exhaustive descriptions of all possible implementations.

Embodiment 1 is directed to an activated soluble carrier comprising an ionotropically crosslinked compound comprising a polymeric material having at least one repeating unit comprising an ionically crosslinked carboxylic acid group and an activated hydroxyl group. , where the hydroxyl group is activated by N,N'-disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidyl chloroformate in a solvent to form a succinimidyl carbonate group for ligand binding. do.

Embodiment 2 is directed to the soluble carrier according to embodiment 1, wherein the carboxylic acid groups are ionically crosslinked with polyvalent cations.

Embodiment 3 is directed to the soluble carrier of embodiment 1, wherein at least one repeating unit comprises:

Embodiment 4 is directed to the soluble carrier according to embodiment 1, wherein the solvent is an aprotic solvent.

Aspect 5 is directed to the soluble carrier of aspect 4, wherein the aprotic solvent is an anhydrous solvent.

Aspect 6 is directed to the soluble carrier of aspect 1, wherein the polymeric material comprises a polygalacturonic acid (PGA) compound.

Aspect 7 is the dissolution according to aspect 1, wherein the PGA compound comprises at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, or a salt thereof. Targeted at sexual carriers.

Embodiment 8 is directed to the soluble carrier according to embodiment 1, wherein the partially esterified pectic acid comprises a degree of esterification from 1 mol% to 40 mol%.

Embodiment 9 is directed to the soluble carrier according to embodiment 1, wherein the partially amidated pectic acid comprises a degree of amidation of from 1 mol% to 40 mol%.

Aspect 10 is directed to the soluble carrier of aspect 1, wherein the soluble carrier comprises a structure comprising beads, fibers, fabrics, foams, or coatings.

Aspect 11 is directed to the soluble carrier of aspect 10, wherein the soluble carrier comprises porous beads.

Aspect 12 is directed to the soluble carrier of aspect 10, wherein the soluble carrier comprises a macroporous foam.

Aspect 13 is directed to the soluble carrier of aspect 10, wherein the soluble carrier comprises a coating for a cell culture surface of a cell culture vessel.

Aspect 14 is directed to the soluble carrier of aspect 1, wherein the soluble carrier is dissolved by digestion with an enzyme, a chelating agent, or a combination thereof.

Aspect 15 is directed to the soluble carrier of Aspect 1, wherein the enzyme comprises a non-proteolytic enzyme.

Aspect 16 is directed to the soluble carrier of Aspect 1, wherein the non-proteolytic enzyme is selected from the group consisting of pectinolytic enzymes and pectinases.

Aspect 17 is directed to the soluble carrier of aspect 1, wherein digestion of the soluble carrier is completed in less than about 1 hour.

Aspect 18 is directed to the soluble carrier of aspect 1, wherein digestion of the soluble carrier is completed in less than about 15 minutes.

Aspect 19 is directed to the soluble carrier of Aspect 1, wherein the ligand comprises a protein, peptide, peptoid, saccharide, or drug.

Aspect 20 is directed to a method of forming an activated soluble carrier comprising forming an ionotropically crosslinked compound. Forming the ionotropically crosslinked compound comprises forming a soluble carrier by adding a polymer solution containing a polygalacturonic acid (PGA) compound to a solution containing at least one polyvalent cation. the PGA compound is selected from at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof; activating the hydroxyl groups to form succinimidyl carbonate groups on the PGA compound of the soluble carrier by adding a oxidizing solution.

Aspect 21 is directed to the method of aspect 20, wherein the method further comprises rinsing the soluble carrier to remove unbound hydroxyl-containing compounds prior to activation.

Aspect 22 is directed to the method of aspect 20, wherein the method further comprises attaching a ligand to the activated hydroxyl group to produce a ligand soluble carrier conjugate.

Aspect 23 is directed to the method of aspect 20, wherein the ligand soluble carrier conjugate comprises at least one unit comprising:

Aspect 24 is directed to the method of aspect 20, wherein the ligand comprises a protein, a peptide, a peptoid, a saccharide, and a drug.

Aspect 25 is directed to the method of Aspect 20, wherein the method further comprises, after activation, rinsing the activated soluble carrier with a solution comprising at least one polyvalent cation.

Aspect 26 is directed to the method of aspect 20, wherein the ionotropically crosslinked compound can be formed into a structure that includes beads, fibers, fabrics, or foams.

Aspect 27 is directed to the method of aspect 20, wherein the ionotropically crosslinked compound can be applied as a coating to a cell culture surface.

Aspect 28 is directed to the method of aspect 20, wherein the activation solution comprises an activator and a solvent.

Aspect 29 is directed to the method of aspect 20, wherein the activating agent comprises N,N'-disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidyl chloroformate.

Aspect 30 is directed to the method of aspect 20, wherein the solvent comprises an aprotic solvent.

Aspect 31 provides that the polygalacturonic acid compound is at least one of pectic acid, partially esterified or amidated pectic acid having a degree of esterification or amidation of 1 to 40 mol%, or a salt thereof. Aspect 20 is directed to a method according to aspect 20, comprising:

Aspect 32 is directed to the method of aspect 20, wherein the polygalacturonic acid compound comprises less than 20 mole % methoxyl groups.

Aspect 33 is directed to a method of culturing cells on a soluble carrier, comprising the steps of seeding the cells on the soluble carrier; and contacting the soluble carrier with a cell culture medium.

Aspect 34 is directed to the method of aspect 33, wherein the step of seeding the cells on the soluble carrier comprises adhering the cells to the surface of the soluble carrier.

Aspect 35 is directed to the method of aspect 33, wherein the cells aggregate within the pores of the soluble carrier to form spheroids.

Aspect 36 is directed to the method of aspect 33, wherein the step of contacting the soluble carrier with the cell culture medium comprises depositing the soluble carrier in the cell culture medium.

Aspect 37 is directed to the method of aspect 33, wherein the step of contacting the soluble carrier with the cell culture medium comprises continuously passing the cell culture medium over the soluble carrier.

Aspect 38 provides that continuously passing the cell culture medium over the soluble carrier removes at least a portion of the cell culture medium from contact with the soluble carrier; 38. A method according to aspect 37, comprising contacting the soluble carrier with fresh cell culture medium such that the volume remains substantially constant.

Embodiment 39 is a method of recovering cells from a soluble carrier, comprising: digesting the soluble carrier by exposing the soluble carrier to an enzyme, a chelating agent, or a combination thereof; collecting the exposed cells when the exposed cells are exposed.

Aspect 40 is an ionotropic carrier, wherein the soluble carrier is selected from at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof. Aspect 39 is directed to the method of aspect 39, wherein the enzyme comprises a polygalacturonic acid compound cross-linked to a polygalacturonic acid compound, and the enzyme comprises a non-proteolytic enzyme.

Aspect 41 is directed to the method of aspect 40, wherein the non-proteolytic enzyme is selected from the group consisting of pectinolytic enzymes and pectinases.

Aspect 42 is directed to the method of aspect 39, wherein digesting the soluble carrier comprises exposing the soluble carrier to between about 1 U and about 200 U of enzyme.

Aspect 43 is directed to the method of aspect 39, wherein digesting the soluble carrier comprises exposing the soluble carrier to between about 1 mM and about 200 mM of a chelating agent.

Although multiple embodiments of the present disclosure are described in the Detailed Description, the present disclosure is not limited to the disclosed embodiments, and may include many other embodiments without departing from the disclosure as described and defined by the claims. It is to be understood that rearrangements, modifications, and substitutions are possible.

Preferred embodiments of the present invention will be described below.

Embodiment 1
an activated soluble carrier comprising:
an ionotropically crosslinked compound comprising a polymeric material having at least one repeating unit, the compound comprising:
an ionically bridged carboxylic acid group, and an activated hydroxyl group, which is activated by N,N'-disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidyl chloroformate in a solvent to bind the ligand. An activated soluble carrier containing hydroxyl groups to form succinimidyl carbonate groups for attachment.

Embodiment 2
The soluble carrier of embodiment 1, wherein the carboxylic acid groups are ionically crosslinked with polyvalent cations.

Embodiment 3
The soluble carrier of embodiment 1, wherein said at least one repeating unit comprises:

Embodiment 4
The soluble carrier according to embodiment 1, wherein the solvent is an aprotic solvent.

Embodiment 5
The soluble carrier according to embodiment 4, wherein the aprotic solvent is an anhydrous solvent.

Embodiment 6
The soluble carrier of embodiment 1, wherein the polymeric material comprises a polygalacturonic acid (PGA) compound.

Embodiment 7
The soluble carrier of embodiment 1, wherein the PGA compound comprises at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, or a salt thereof. .

Embodiment 8
A soluble carrier according to embodiment 1, wherein the partially esterified pectic acid comprises a degree of esterification of 1 mol% to 40 mol%.

Embodiment 9
A soluble carrier according to embodiment 1, wherein the partially amidated pectic acid comprises a degree of amidation of 1 mol% to 40 mol%.

Embodiment 10
The soluble carrier of embodiment 1, wherein the soluble carrier comprises a structure comprising beads, fibers, fabrics, foams, or coatings.

Embodiment 11
11. The soluble carrier of embodiment 10, wherein the soluble carrier comprises porous beads.

Embodiment 12
11. The soluble carrier of embodiment 10, wherein the soluble carrier comprises a macroporous foam.

Embodiment 13
11. The soluble carrier of embodiment 10, wherein the soluble carrier comprises a coating for a cell culture surface of a cell culture vessel.

Embodiment 14
The soluble carrier of embodiment 1, wherein the soluble carrier is dissolved by digestion with an enzyme, a chelating agent, or a combination thereof.

Embodiment 15
The soluble carrier of embodiment 1, wherein the enzyme comprises a non-proteolytic enzyme.

Embodiment 16
The soluble carrier according to embodiment 1, wherein the non-proteolytic enzyme is selected from the group consisting of pectinolytic enzymes and pectinases.

Embodiment 17
The soluble carrier of embodiment 1, wherein digestion of the soluble carrier is completed in less than about 1 hour.

Embodiment 18
The soluble carrier of embodiment 1, wherein digestion of the soluble carrier is completed in less than about 15 minutes.

Embodiment 19
The soluble carrier of embodiment 1, wherein the ligand comprises a protein, peptide, peptoid, saccharide, or drug.

Embodiment 20
1. A method of forming an activated soluble carrier comprising:
forming an ionotropically crosslinked compound, the step comprising:
forming a soluble carrier by adding a polymer solution containing a polygalacturonic acid (PGA) compound to a solution containing at least one polyvalent cation, wherein the PGA compound is pectic acid, partially esterified; activating the hydroxyl groups by forming and adding an activation solution selected from at least one of pectic acid, partially amidated pectic acid, and salts thereof to forming a succinimidyl carbonate group on said PGA compound of a sexual carrier.

Embodiment 21
21. The method of embodiment 20, wherein the method further comprises rinsing the soluble carrier to remove unbound hydroxyl-containing compounds prior to activation.

Embodiment 22
21. The method of embodiment 20, wherein the method further comprises attaching a ligand to the activated hydroxyl group to produce a ligand soluble carrier conjugate.

Embodiment 23
The method of embodiment 20, wherein the ligand soluble carrier conjugate comprises at least one unit comprising:

Embodiment 24
21. The method of embodiment 20, wherein the ligands include proteins, peptides, peptoids, saccharides, and drugs.

Embodiment 25
21. The method of embodiment 20, wherein the method further comprises, after activation, rinsing the activated soluble carrier with a solution comprising at least one multivalent cation.

Embodiment 26
21. The method of embodiment 20, wherein the ionotropically crosslinked compound can be formed into structures including beads, fibers, fabrics, or foams.

Embodiment 27
21. The method of embodiment 20, wherein the ionotropically crosslinked compound can be applied as a coating to a cell culture surface.

Embodiment 28
21. The method of embodiment 20, wherein the activation solution includes an activator and a solvent.

Embodiment 29
21. The method of embodiment 20, wherein the activator comprises N,N'-disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidyl chloroformate.

Embodiment 30
21. The method of embodiment 20, wherein the solvent comprises an aprotic solvent.

Embodiment 31
The polygalacturonic acid compound comprises at least one of pectic acid, partially esterified or amidated pectic acid having a degree of esterification or amidation of 1 to 40 mol%, or a salt thereof. , the method of embodiment 20.

Embodiment 32
21. The method of embodiment 20, wherein the polygalacturonic acid compound contains less than 20 mole % methoxyl groups.

Embodiment 33
A method of culturing cells on a soluble carrier, the method comprising:
A method comprising: seeding cells on a soluble carrier; and contacting the soluble carrier with a cell culture medium.

Embodiment 34
34. The method of embodiment 33, wherein the step of seeding cells on a soluble carrier comprises adhering cells to the surface of the soluble carrier.

Embodiment 35
34. The method of embodiment 33, wherein cells aggregate within the pores of the soluble carrier to form spheroids.

Embodiment 36
34. The method of embodiment 33, wherein the step of contacting the soluble carrier with a cell culture medium comprises depositing the soluble carrier in a cell culture medium.

Embodiment 37
34. The method of embodiment 33, wherein contacting the soluble carrier with cell culture medium comprises continuously passing cell culture medium over the soluble carrier.

Embodiment 38
continuously passing cell culture medium over the soluble carrier, removing at least a portion of the cell culture medium from contact with the soluble carrier; and cell culture medium in contact with the soluble carrier. 38. The method of embodiment 37, comprising contacting the soluble carrier with fresh cell culture medium such that the volume of the soluble carrier is maintained substantially constant.

Embodiment 39
A method for recovering cells from a soluble carrier, the method comprising:
digesting the soluble carrier by exposing the soluble carrier to an enzyme, a chelating agent, or a combination thereof; and collecting exposed cells when the soluble carrier is digested. Method.

Embodiment 40
The soluble carrier is ionotropically cross-linked selected from at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, and salts thereof. 40. The method of embodiment 39, wherein the enzyme comprises a non-proteolytic enzyme.

Embodiment 41
41. The method of embodiment 40, wherein the non-proteolytic enzyme is selected from the group consisting of pectinolytic enzymes and pectinases.

Embodiment 42
40. The method of embodiment 39, wherein digesting the soluble carrier comprises exposing the soluble carrier to between about 1 U and about 200 U of the enzyme.

Embodiment 43
40. The method of embodiment 39, wherein digesting the soluble carrier comprises exposing the soluble carrier to between about 1 mM and about 200 mM of the chelating agent.

Claims (15)

an activated soluble carrier comprising:
an ionotropically crosslinked compound comprising a polymeric material having at least one repeating unit, the compound comprising:
an ionically bridged carboxylic acid group, and an activated hydroxyl group, which is activated by N,N'-disuccinimidyl carbonate (DSC) or N-hydroxysuccinimidyl chloroformate in a solvent to bind the ligand. An activated soluble carrier containing hydroxyl groups to form succinimidyl carbonate groups for attachment. The soluble carrier according to claim 1, wherein the carboxylic acid groups are ionically crosslinked with polyvalent cations. The soluble carrier of claim 1, wherein the at least one repeating unit comprises:

The soluble carrier according to claim 1, wherein the solvent is an aprotic solvent. The soluble carrier of claim 1, wherein the polymeric material comprises a polygalacturonic acid (PGA) compound. 6. The soluble carrier of claim 5, wherein the PGA compound comprises at least one of pectic acid, partially esterified pectic acid, partially amidated pectic acid, or a salt thereof. . A soluble carrier according to claim 6, wherein the partially esterified pectic acid comprises a degree of esterification of 1 mol% to 40 mol%. A soluble carrier according to claim 7, wherein the partially amidated pectic acid comprises a degree of amidation of 1 mol% to 40 mol%. 2. The soluble carrier of claim 1, wherein the soluble carrier comprises a structure comprising beads, fibers, fabrics, foams, or coatings. 1. A method of forming an activated soluble carrier comprising:
forming an ionotropically crosslinked compound, the step comprising:
forming a soluble carrier by adding a polymer solution containing a polygalacturonic acid (PGA) compound to a solution containing at least one polyvalent cation, wherein the PGA compound is pectic acid, partially esterified; activating the hydroxyl groups by forming and adding an activation solution selected from at least one of pectic acid, partially amidated pectic acid, and salts thereof to forming a succinimidyl carbonate group on said PGA compound of a sexual carrier. 11. The method of claim 10, wherein the method further comprises rinsing the soluble carrier to remove unbound hydroxyl-containing compounds prior to activation. 11. The method of claim 10, wherein the method further comprises attaching a ligand to the activated hydroxyl group to produce a ligand-soluble carrier conjugate. 13. The method of claim 12, wherein the ligand soluble carrier conjugate comprises at least one unit comprising:

13. The method of claim 12, wherein the ligands include proteins, peptides, peptoids, saccharides, and drugs. 11. The method of claim 10, wherein the method further comprises, after activation, rinsing the activated soluble carrier with a solution comprising at least one polyvalent cation.

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